Isolation of Culture-Difficult Gram-Negative Coccobacilli

Objective: We have made efforts to isolate so-called culture-difficult oral bacteria, and some of those belonging to assacharolytic anaerobic Gram-positive rods (AAGPR) were successfully cultured and reported as new bacterial genus/species. The present study aimed to isolate and culture assacharolytic anaerobic Gram-negative coccobacilli (AAGNC) that were more difficult than AAGPR. Methods: Bacteria in periodontal pockets of more than 6 mm depth were collected using micropipettes under anaerobic gas atmosphere, transferred in tight capped vials filled with anaerobic gas, diluted and cultured in an anaerobic glove box that was maintained under strictly anaerobic conditions. A stereoscope equipped to the anaerobic glove box was sometimes necessary for isolation/culture procedures. Results: Of 9 samples, 3 were positive for AAGNC at whole 0.5-1.5%, while 8 contained AAGPR at 0.8-27%. AAGNC colonies on BHI-blood agar plates were less than 0.2 mm diameter, and the organisms were coccoids or short rods of 0.2-0.5 X 0.7 µm. A total of 10 isolates were grouped to those producing malate (0.4-0.8 mM, 2 isolates) and propionate (0.3-0.4 mM, 8 isolates), respectively, as an end product. They all were inert in all biochemical examinations tested. Using 16S rDNA sequence and GC-content analyses, the former isolates were found to resemble closely to Dialister pneumosintes (2 isolates). The latter 7 isolates were identified as D. invisus, and one closely resembled to D. invisus. Conclusion: AAGNC, together with AAGPR seemed to be predominant in bacterial florae of periodontal pockets. The efforts to isolate culture-difficult bacteria may contribute for understanding of their pathogenic characteristics, and for increased bacterial gene information for DNA analyses, which could be possible only using living isolates.